PROJECT SUMMARY Glioblastoma multiforme (GBM) is the most common and aggressive primary brain tumor, with an extremely poor prognosis. Standard therapies fail to eradicate residual or infiltrating cells that reside adjacent to and infiltrate normal brain tissue. Stem cells are emerging as feasible delivery vehicles to therapeutically target primary and invasive tumor cells. One shortcoming of this approach of using stem cells as therapeutic vehicles is that in addition to migrating towards tumors, stem cells are additionally attracted towards normal areas in the body that may be harmed if they non-selectively express highly toxic therapies. Therefore, the objective of our application is to create a remote-controllable stem cell-based strategy that will allow us to non-invasively activate stem cell therapeutic production at the time and place of our choosing. The central hypothesis to be tested is that image-guided high intensity focused ultrasound (HIFU) can be used in an innovative way to mildly heat tumor tissue at the depth of our choosing and induce therapeutic production controlled by the HSP70 promoter. Of translational relevance, technology that delivers HIFU through the human skull to a depth of the operator's choosing is already being used in clinic for other applications. Another major limitation of cell-based therapy is the number of cells that successfully navigate to the tumor site. We propose to use HIFU to drive the production of a pro-migratory factor, which should further increase stem cell trafficking and accumulation at the tumor site. This process, the controlled amplification of stem-cell trafficking (CAST), may be repeated to serially amplify the effect. Since CAST is controlled by physical rather than biological stimuli, it can avoid the tumor immuno-suppression that cripples traditional cell therapy. Our remote controlled expression platform can also be leveraged to assist in locally opening up the blood brain barrier for facilitated drug delivery. The opening of the BBB will be limited to where selected factors will be secreted secondary to HIFU activation, which is in the vicinity of the engineered stem cells. As a result even though a much larger volume will be heated by HIFU the blood brain barrier opening will be much more focused to where the stem cells are located. If successful, this degree of spatial and temporal precision in controlling the blood brain barrier opening will be unprecedented and can have great potential in applications even outside of brain tumor therapy. To test our hypothesis we will pursue three specific aims: (1) To demonstrate that image-guided HIFU-mediated remote heat-activation of engineered stem cells can non-invasively activate expression of reporter genes; (2) To demonstrate the feasibility of using image-guided pHIFU to induce anti-GBM therapeutics under control of the HSP70 promoter; and (3) To determine the feasibility of using image-guided HIFU to temporally induce cytokine expression (under control of HSP70 promoter) for the purpose of attracting a second amplified wave of therapeutic stem cells to the target site and focally permeabilizing the Blood Brain Barrier (BBB).